Proposal for an “Althaeae flos (marshmallow flower ... · Acta Pharmaceutica Sciencia. Vol. 56 No. 1, 201837 Proposal for an “Althaeae flos (marshmallow flower)” Monograph set-up

37Acta Pharmaceutica Sciencia. Vol. 56 No. 1, 2018

Proposal for an “Althaeae flos (marshmallow flower)” Monograph set-up based on European Pharmacopoeia Template and Quality Evaluation of Commercial Samples

Acta Pharm. Sci. Vol 56 No: 1. 2018DOI: 10.23893/1307-2080.APS.05603

Esra Saçıcı1*, Erdem Yeşilada1

1Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, İstanbul, Turkey

*Corresponding author: Esra Saçıcı, e-mail: [email protected](Received 16 November 2017, accepted 22 November 2017)

INTRODUCTION

There are 4 species represent of Althaea L. genus (Malvaceae) in “The Flora of Turkey and the East Aegean Islands”.1 Among these species, Althaea officinalis L. (marshmallow) is indigenous to Turkish flora and it is native in Asia, Europe and China.2,3 In Turkey, A. officinalis is commonly named as “hatmi, tıbbi hatmi, devegülü, fatma çiçeği, erkurtaran/ergurtaran çiçeği”.4-7

The name of Althaea genus is derived from a word “althaino” that signifies “ther-apy, treatment” in Greek because of the healing potential of the plant and the species name “officinalis” signifies “to use in medicine”.8

ABSTRACT

Although marshmallow flowers are used more frequently than leaves and roots, only Althaeae folium and Althaeae radix monographs exist in European Pharma-copoeia. The aim of study was to set standards for marshmallow flowers which will be used for its quality assessment. Althaeae flos monograph was prepared based on the main principles described in European Pharmacopoeia for herbal drugs. After setting the limits, the specifications of 33 marshmallow samples purchased from herbal markets were studied to determine their suitability for the proposed mono-graph. Results have shown that all the commercial samples were obtained not from Althaea, but Alcea species, a close gender, based on the flowers size. Since main characteristics of both flowers were determined to be identical, this would not rise a problem. However, residual parts of insects or snails were found in 28 samples and thus they were evaluated unacceptable to be used in phytotherapy.Keywords: Althaeae flos, marshmallow flower, Malvaceae, monograph, quality

38 Acta Pharmaceutica Sciencia. Vol. 56 No. 1, 2018

According to the studies on chemical constituents, astragalin9, 4-hydroxy-ben-zoic acid10, ferulic acid10, hypolaetin 8-0-gentiobioside11, hypolaetin-4-0-me-thyl ether-8-0-β-D-glucoside11, dihydrokaempferol 4-0-β-D-glucoside11, dihy-drokaempferol 4-0-glucoside9, naringenin-4-0-β-D-glucoside11, naringenin-4-0-glucoside12, p-hydroxy-phenyl acetic acid13,14, populnin10, protocatechuic acid14, isoquercitrin10, salicyclic acid13, scopoletin14, sinapic acid14, spiraeoside12, syringic acid13, tiliroside15 and also vanillic acid10 were isolated from the flower parts of A. officinalis.

In vitro antioxidant effect of the flower extract from A. officinalis was investi-gated and reported to possess strong antioxidant effect, i.e., significant metal chelating, superoxide anion radical scavenging, free radical scavenging and re-ducing power actions.16 In another study, the total flavonoids content and the antioxidant properties of Althaeae flos were comparatively determined in terms of the petal colors, including white, pink and reddish pink. Although white petals possessed the highest flavonoid content, reddish-pink colored petals exhibited the highest antioxidant activity.17

In a study, possible role of the aqueous extract of Althaeae flos in platelet ag-gregation, lipemia, inflammation and gastric ulcer was examined in rats. It was reported that platelet aggregation was inhibited time-dependently. Serum HDL-cholesterol level was significantly increased, while no effects on stool cho-lesterol and triacylglycerol were observed. The aqueous extract also showed anti-inflammatory activity on acute and chronic inflammation models and also anti-ulcerogenic effect was reported without any apparent adverse effect.18 In another study, hexane extract of Althaeae flos collected from northwestern Iran was found to contain fatty acid composed of alpha-linolenic acid, palmitic acid, heptacosane and nonacosane, and the extract displayed a good antimicrobial activity against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Candida albicans and Saccharomyces cerevisiae.19

Roots and leaves of Althaea species have been used in traditional medicines worldwide for centuries as expectorant, demulcent, diuretic, and emollient for the treatment of cough, upper respiratory tract inflammations, gastritis, pep-tic ulceration, renal (i.e. cystitis) and uterine disorders.20-23 However records for use of its flowers in traditional medicines are rare. Hot water extract prepared from the dried marshmallow flowers was utilized externally (in Peru)24 or orally (in France)25 as an emollient or as expectorant in India.26 Additionally, decoc-tions prepared from the flowers and leaves were taken orally against asthmatic symptoms in Italy.27 In Greece, root and flowers of A. officinalis have found a wide range of applications including coronary disorders, hypertension, consti-


pation, diarrhea, enteritis, gastritis, stomatitis, cystitis, kidney stones, nephritis, blood purification, laryngitis, pharyngitis, common cold, bronchitis, aphthae, gum wounds and pimples.28 In the Iranian traditional documents, the flowers, leaves or roots of A. officinalis have all been suggested orally for gastrointestinal and respiratory system inflammations as well as externally for burns and in-sect stings.29 Furthermore, as an evidence of safety of A. officinalis, it is listed by the Council of Europe in category N2 as a natural food as well as in the United States. 22,30,31

In Turkey, infusions obtained from marshmallow flowers have been used in-ternally for bronchial complaints, cough and cold due to its rich mucilage con-tent.5,32 Decoction of marshmallow root have been used internally for the treat-ment of gastritis, peptic, mouth and throat ulcerations as well as for respiratory tract irritations as gargle.4 In East Anatolia, infusion obtained from A. officinalis aerial parts has been used as diuretic and antilithic in the treatment of kidney stone or sand.33 It is reported that in Aydıncık district (Mersin), infusion pre-pared from A. officinalis herb has also been used orally as one teacup 3 times per day for 2-3 weeks or used as gargle for 3-5 successive days against stomachache, as well as asthma, cold and flu symptoms.7 [Note: In both studies the plant part used as remedy was specified as “herb”]. Moreover, Althaea roots are externally applied on wounds to keep the skin soft and for wound-healing.4

Despite A. officinalis flowers have found a place in traditional medicinal practice, in the European Pharmacopoeia (Ph. Eur.), only the qualifications of its roots and leaves are described under two monographs; Marshmallow leaf (Althaeae folium)34 and Marshmallow root (Althaeae radix)35. Although in French Phar-macopoeia the flowers were registered together with the roots and leaves as a herbal drug for the treatment of cough, but no specification was described except the common macroscopic and microscopic characteristics.36 On the other hand, in Turkey only the dried flowers of the plant have been available in the herbal drug market, the official herbal drugs, i.e., roots or leaves of marshmallow, are not available. Eventually, there is no official data to describe the specifications of marshmallow flowers to determine its qualification. Therefore, the first aim of the present study was to define the macroscopical, microscopical and chemical characteristics of marshmallow flowers. Secondly, a draft monograph proposal for “Althaeae flos (marshmallow flower)” was suggested to set the standards for the herbal drug by predicating on the analysis criteria in the Ph. Eur. In com-pliance with the proposed template, quality evaluation was carried out on the marshmallow flower samples purchased from herbal shops.


MATERIALS AND METHODS

Plant materials and solvents

Thirty-three samples of marshmallow flowers were purchased from local herb markets in İstanbul and Tekirdağ (Turkey). The other herbal material Alcea sp. flowers collected from Karaman, Konya (Turkey) in order to be used in this work and they were dried in shade. In this study, specimens of marshmallow were encoded as A1-A33 throughout the study for reasons of confidentiality. All of these herbs were powdered by a laboratory mill. Ethanol and sulphuric acid were from Sigma-Aldrich (Steinheim, Germany); acetic acid, n-butanol and methanol were purchased from Riedel-de Haën (Seelze, Germany), Fluka (Steinheim, Ger-many) and Analar Normapur (Muarrie, Australia), respectively.

Methods

Macroscopic examination

The morphological characteristics of the whole materials were determined un-der the Stems DV4/DR Carl Zeiss binocular loop. Epicalyx, calyx, corolla, sta-men, filament, anther, ovary, stylus and pedicel parts of these materials were evaluated. In addition, the petal lengths of marshmallow flowers and Alcea sp. flowers were measured by a ruler.

Microscopic examination

The microscopic properties of the powdered materials were determined under a Carl Zeiss AxioVision electronical microscope and a Nikon E200 microscope using chloral hydrate solution. Characteristic elements i.e. curved, stellate or glandular trichomes, calcium oxalate crystals, pollen grains and stomata were examined.

Detection of foreign matter

The amounts of foreign organs and foreign elements of the entire flower ma-terials were calculated according to the technique for determination of foreign matter described under Methods in Pharmacognosy in the Ph. Eur. 50-100 g of the whole samples were weighed. Then, they were spread on a white paper and insects, snails, feathers, bristles and stones were separated by examining with unaided eye as well as a magnifying glass. The removed foreign materials were simply weighed and the percentages were calculated.

Loss on drying experiment

The quantities of losses on drying of all the samples were evaluated according to technique for loss on drying under Physical and Physicochemical Methods in


the Ph. Eur. 1 g of the whole material was weighed in capsules, and dried in the drying oven at 105 °C for 2 hours. Then, capsules were cooled, reweighed, and the percentages were calculated.

Total ash experiment

The ash amount of each material was determined according to the method de-scribed under Limit Tests in the Ph. Eur. for total ash. All the crucibles were heated for 30 minutes, cooled in a desiccator and tares of them were weighed. 1 g of the powdered substance was weighed in a crucible, and then was ignited in a furnace at 600 °C. After each burning, the crucible was removed from furnace, cooled in a desiccator and weighed when it was cool and then the total ash per-centage was calculated.

Swelling index experiment

The swelling index of all samples were calculated according to the method de-scribed under Methods in Pharmacognosy in the Ph. Eur. 0.2 g of each of the powdered material was weighed and put in a 25 mL ground-glass stoppered spe-cial cylinder graduated in 0.5 mL divisions. The material was first humidified with 0.5 mL of ethanol, and 25 mL of distilled water was added. Then each glass cylinder was enclosed and shaken every ten minutes for one hour period, and left to stand for three hours. After 90 minutes from the start of the experiment, the cylinders were turned around their axes in order to mix large volumes of liquid retained in the layer of the drugs and fragments of these drugs floating on the top of the liquid. At the end of the test, the volume occupied by the swelling drug, which indicates mucilage content, was measured. Three individual experiments were performed for each material at the same time. The average of three trials was estimated as the swelling index.

High Performance Thin-Layer Chromatography (HPTLC) analysis

The analyses were carried out on alumina HPTLC plates covered with silica gel 60 F254 (Merck, Darmstadt, Germany). 1 g of each powdered material was weighed, and 10 mL of 60% ethanol was added. After stirring for 15 min, they were filtered to prepare test solutions. These solutions were applied on the plates as 9.0 mm bands by a mean of 100 µL syringe (Hamilton, Bonaduz, Switzerland) via Camag Linomat V sample applicator (Muttenz, Switzerland). The plates were developed in a twin through glass chamber saturated with the mobile phase. The same mobile system [acetic acid-water-butanol (15:30:60, v/v/v)] recorded in “Malvae sylvestris flos” monograph in Ph. Eur. was used. After developing the plates, they were dried. Chromatograms were displayed by exposing to UV-light at 366 nm using Camag TLC Visualizer. Then, the plates were sprayed with sul-


phuric acid 5% in methanol (H2SO4) reagent. The derivatized plates were heated at 105 °C and documented. All the devices were managed by WinCATS software.

RESULTS

The findings of thirty-three marshmallow samples and Alcea sp. materials were evaluated by using the data presented under “Malvae sylvestris flos” monograph in the Ph. Eur.37 This monograph was used as a guiding reference because Malva sylvestris is a close member to Althaea sp. from the same family.

Macroscopic results

Macroscopic examinations of all commercial marshmallow materials demon-strated that they had similar morphological features to Alcea sp. On the other hand, petal lengths of all these marshmallow materials were not within the limits specified for Althaea L. in “The Flora of Turkey and the East Aegean Islands”, but they were similar to that of the Alcea sp. (Table 1). The morphological find-ings obtained by macroscopic analyses are given in Table 2.

Table 1. Comparative macroscopic characteristics of Althaea L. and Alcea L.38,39

Alcea L. Althaea L.

Epicalyx generally 6, rarely 7-9 partiteconnate

6-9 partiteconnate at the base

Petal 30 mm or longer up to 16 mm

Anther yellowish brownish-purple

Carpel sub-bilocular unilocular


Tab

le 2

. Obs

erve

d fin

ding

s fro

m th

e m

acro

scop

ic a

naly

sis

of c

omm

erci

al m

arsh

mal

low

flow

er s

ampl

es

A-1,

A-7

, A-8

, A-9

, A-1

3, A

-19,

A-2

3, A

-24,

A-2

5, A

-26,

A-

27, A

-28

6 pa

rtite

st

ella

te tr

icho

me

stel

late

tric

hom

e5

sepa

ls

A-13

. pur

ple,

whi

teA-

19, 2

3, 2

4, 2

5: p

ink,

pur

ple

A-26

: lig

ht b

lue,

ligh

t pur

ple

A-27

: dar

k or

ange

, pur

ple

num

erou

s

stel

late

tric

hom

e

dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

-21-4

9 m

mA-

1, 9

, 24:

long

er th

an c

alyx

A-7,

8, 2

8: p

urpl

eA-

9: p

ink,

pur

ple,

red

A-15

& A

-33

5-6

part

ite

stel

late

tric

hom

e

stel

late

tric

hom

e5

sepa

ls

20-3

2 m

mpu

rple

num

erou

s

stel

late

tric

hom

e

dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

A-33

: ste

llate

tric

hom

e

A-3

& A

-21

5 pa

rtite

st

ella

te tr

icho

me

stel

late

tric

hom

eA-

3: 5

sep

als

A-21

: 6 s

epal

s

25-4

4 m

m

A-3:

pin

kA-

21: p

urpl

e

num

erou

s

stel

late

tric

hom

e

dors

ifix

A-21

: gen

eral

ly s

pille

d

herm

aphr

odite

2-lo

cula

r

num

erou

s

-

Alce

a sp

.

6 pa

rtite

st

ella

te tr

icho

me

stel

late

tric

hom

e5

sepa

ls

16-3

2 m

mw

hite

num

erou

s

- dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

stel

late

tric

hom

e

Ep

ical

yx

Cal

yx

Co

rolla

Sta

men

Fila

men

t

An

ther

Flo

wer

Ova

ry

Sty

lus

Ped

icel


Tab

le 2

. Obs

erve

d fin

ding

s fro

m th

e m

acro

scop

ic a

naly

sis

of c

omm

erci

al m

arsh

mal

low

flow

er s

ampl

es (c

ontin

ued)

A-1

1

7-8

parti

te

stel

late

tric

hom

e

stel

late

tric

hom

e5

sepa

ls

yello

w, li

ght p

ink,

pur

ple

32-4

5 m

m

num

erou

s

stel

late

tric

hom

e

dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

-

A-6

, A-1

0, A

-17,

A-1

8, A

-20,

A

-32

7 pa

rtite

st

ella

te tr

icho

me

stel

late

tric

hom

eA-

6, 1

7, 2

0, 3

2: 5

sep

als

A-10

, 18:

6 s

epal

s

24-5

1 m

mA-

6: lo

nger

than

cal

yxA-

6, 1

0, 2

0: p

urpl

eA-

17: p

ink,

pur

ple

A-18

: dar

k pi

nk, r

ed, p

urpl

e, p

ink

A-32

: lig

ht p

ink,

dar

k pu

rple

num

erou

s

stel

late

tric

hom

e

dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

-

A-2

, A-1

4, A

31

6-7

parti

te

stel

late

tric

hom

e

stel

late

tric

hom

eA-

2 &

A-3

1: 5

sep

als

A-14

: 3 s

epal

s

17-4

3 m

mA-

14: p

ink,

pur

ple

A-31

: dar

k or

ange

, pur

ple

num

erou

s

stel

late

tric

hom

e

dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

-

A-5

, A-1

2, A

-16,

A-2

2,

A-2

9, A

-30

6 pa

rtite

st

ella

te tr

icho

me

stel

late

tric

hom

eA-

5: 6

sep

als

A-12

, 16,

22,

29:

5 s

epal

sA-

30: 3

-5 s

epal

s

22-5

3 m

mA-

5: li

ght &

dar

k pi

nkA-

12, 2

2, 2

9: p

urpl

eA-

16: y

ello

wis

hA-

30: l

ight

& d

ark

purp

le

num

erou

s

stel

late

tric

hom

e

dors

ifix

herm

aphr

odite

2-lo

cula

r

num

erou

s

stel

late

tric

hom

e

A-4

6 pa

rtite

st

ella

te tr

icho

me

stel

late

tric

hom

e5

sepa

ls

29-3

1 m

m

dark

pin

k

num

erou

s

stel

late

tric

hom

e

- herm

aphr

odite

2-lo

cula

r

num

erou

s

-

Ep

ical

yx

Cal

yx

Co

rolla

Sta

men

Fila

men

t

An

ther

Flo

wer

Ova

ry

Sty

lus

Ped

icel


Results of Microscopic Analysis

The microscopic analysis of all commercial marshmallow samples exhibited that they had similar microscopic characteristics to Alcea sp. (Figure 1). Additionally, stomata of A-2, 5, 10, 22, 32 and 33 had 3 subsidiary cells as well as only A-3 had the Malvaceae type glandular trichome. The microscopic findings are presented in Table 3.

Figure 1. Microscopic illustrations of Alcea sp.: (a) stellate trichome (4x); (b) curved tri-chome (40x); (c) stomata (40x); (d) glandular trichome (40x); (e) cluster crystals of calcium oxalate (40x); (f) vessel (40x); (g) petal epidermis (40x); (h) glandular trichome in petal (40x); (i) pollen grain (40x).


Tab

le 3

. Com

para

tive

eval

uatio

n of

the

findi

ngs

from

mic

rosc

opic

al a

naly

sis

A-23

, A-2

7, A

-28,

A-3

1

4-8

part

ite

+ - + + num

erou

ssm

all &

big

-

A-1,

A-2

5

A-1:

4-8

par

tite

A-25

: 4-7

par

tite

+ - - + num

erou

ssm

all &

big

-

A-8,

A-9

, A-1

3, A

-14,

A-1

5

3-9

part

ite

+ num

erou

s

- A-8,

9, 1

5: +

A-13

, 14:

-

+ num

erou

ssm

all &

big

-

A-2,

A-5

, A-1

0, A

-22,

A-

32, A

-33

A-2:

3-9

par

tite

A-5:

4-7

par

tite

A-22

, 32:

4-9

par

tite

A-10

: 5-7

par

tite

A-33

: 6-9

par

tite

+ - A-2,

5, 1

0, 3

2, 3

3: +

A-22

: -

+ num

erou

ssm

all &

big

+ 3 su

bsid

iary

cel

ls

A-7

3-8

part

ite

+ num

erou

s

- + + num

erou

ssm

all

-

Alce

a sp

.

5-9

part

ite

+ num

erou

s

+ + + num

erou

ssm

all &

big

+ 3-5

subs

idia

ry c

ells

Ste

llate

tr

ich

om

e

Cu

rved

tr

ich

om

e

Gla

nd

ula

r tr

ich

om

e

Cal

ciu

m

oxa

late

cr

ysta

l

Po

llen

gra

in

Po

llen

gra

in


Tab

le 3

. Com

para

tive

eval

uatio

n of

the

findi

ngs

from

mic

rosc

opic

al a

naly

sis

(con

tinue

d)

A-1

2, A

-18,

A-1

9

6-9

part

ite

+ - + + num

erou

ssm

all &

big

-

A-2

0

6-8

part

ite

+ - + + num

erou

ssm

all &

big

-

A-6

, A-2

9

5-9

part

ite

+ - + + num

erou

ssm

all &

big

-

A-3

, A-1

1, A

-16,

A-1

7,

A-2

1, A

-24

5-8

part

ite

+ A-3:

+A-

11, 1

6, 1

7, 2

1, 2

4: -

A-3,

11,

17,

24:

+A-

16, 2

1: -

+ num

erou

ssm

all &

big

-

A-2

6

5-7

part

ite

+ num

erou

s

- + + num

erou

ssm

all &

big

-

A-4

4-9

part

ite

+ num

erou

s

- - + num

erou

ssm

all &

big

-

Ste

llate

tr

ich

om

e

Cu

rved

tr

ich

om

e

Gla

nd

ula

r tr

ich

om

e

Cal

ciu

m

oxa

late

cr

ysta

l

Po

llen

gra

in

Po

llen

gra

in


Foreign Matter

The ratio of foreign matter is set at maximum 2.0 per cent (w/w) as described at the general pharmacopoeia rules. Accordingly, 27 of 33 commercial marsh-mallow specimens were found to be lower than this ratio, while the percentages of the remaining six marshmallow materials [A-3, 16, 22, 27, 30, and 31] were over 2.0% (Figure 2). On the other hand, insects or insect parts and snails were detected in 26 among the 33 marshmallow samples, therefore they cannot be considered as safe herbal drugs. Among the remaining seven samples which did not contain insects and snails, two samples were previously eliminated due to high ratio of foreign matter. Consequently, only five samples [A-11, 12, 13, 18, and 19] were considered to be appropriate herbal drug. The percentages of for-eign organs and foreign elements are given in Table 4.

Figure 2. Foreign substances detected in marshmallow samples. (a) feather & different plant parts in A-3; (b) feather, bristle & different plant parts in A-7; (c) different plant parts in A-13; (d) stone & different plant parts in A-15; (e) different plant parts in A-16; (f) stone & different plant parts in A-20; (g) insects in A-21; (h) insects & snail in A-23; (i) insects in A-24; (j) different plant parts in A-29; (k) feather & different plant parts in A-30; (l) insect in A-32.


Table 4. Findings of foreign matter

Samples Insects/Snails(%)

Feathers/Bristles/Stones/Foreign plant parts

(%)

Total foreign matter

(%)

A-1 0.029 – 0.029

A-2 1.526 0.440 1.966

A-3 – 5.696 5.696

A-4 0.399 – 0.399

A-5 0.023 0.303 0.326

A-6 0.021 0.055 0.076

A-7 0.017 0.264 0.281

A-8 0.018 0.311 0.329

A-9 0.028 0.007 0.035

A-10 0.109 0.612 0.721

A-11 – 0.857 0.857

A-12 – 0.038 0.038

A-13 – 0.903 0.903

A-14 0.006 0.212 0.218

A-15 0.017 0.300 0.317

A-16 – 2.792 2.792

A-17 1.529 – 1.529

A-18 – 0.843 0.843

A-19 – 0.158 0.158

A-20 0.695 0.498 1.193

A-21 0.235 0.215 0.450

A-22 2.169 – 2.169

A-23 0.239 0.429 0.668

A-24 0.285 0.123 0.408

A-25 0.010 0.077 0.087

A-26 0.003 1.994 1.997

A-27 0.051 4.336 4.387

A-28 0.015 0.940 0.955

A-29 0.064 0.410 0.474

A-30 0.007 2.363 2.370

A-31 0.069 2.618 2.687

A-32 0.021 1.264 1.285

A-33 0.101 0.641 0.742


Loss on Drying

The limit of loss on drying was set at maximum 12.0 per cent (w/w) similarly to set value in “Malvae sylvestris flos” monograph [Ph. Eur.]. Accordingly, in 28 samples the rates were found to be lower [between 9.74 and 11.94 for A-1 to A-4, A-6 to A-16, A-18 to A-20, A-22, A-23, and A-26 to A-33], while in five samples [A-5, 17, 21, 24, 25] and reference Alcea sp. the ratio was higher than 12.0% [be-tween 12.28 and 13.25].

Total Ash

In the “Malvae sylvestris flos” monograph total ash limit was set at maximum 14.0 per cent (w/w). Accordingly, the total ash amount of all the commercial marshmallow samples [between 8.36 and 11.63 for A-1, A-2, A-4 to A-33] and Alcea sp. sample have remained below this limit. The ash content of only A-3 was somewhat closer to 14.0% [13.52%].

Swelling Index

Swelling index was determined following the “Malvae sylvestris flos” monograph [Ph. Eur.] and the limit therein was at least 15. In this regard, the swelling indi-ces of all marshmallow specimens and Alcea sp. sample were far below the limit value. Besides, the greatest swelling indices were obtained from only A-24 and A-25. The swelling indices are presented in Table 5.


Table 5. Findings of swelling index

Samples Trial-I Trial-II Trial-III Mean

Alcea sp. 7.25 7.00 6.50 6.92

A-1 3.00 4.25 3.00 3.42

A-2 3.50 5.50 3.50 4.17

A-3 6.00 8.50 7.50 7.33

A-4 6.00 4.50 5.50 5.33

A-5 6.00 8.50 7.00 7.17

A-6 4.50 4.00 4.00 4.17

A-7 4.00 4.50 3.50 4.00

A-8 4.50 4.00 4.50 4.33

A-9 4.50 5.00 4.50 4.67

A-10 3.50 4.00 3.00 3.50

A-11 5.50 5.00 4.50 5.00

A-12 7.00 5.00 5.00 5.67

A-13 5.50 6.00 5.00 5.50

A-14 4.50 3.00 3.50 3.67

A-15 5.00 5.50 6.50 5.67

A-16 3.00 3.00 2.25 2.75

A-17 4.50 6.00 4.50 5.00

A-18 4.00 5.50 5.00 4.83

A-19 4.00 4.50 4.50 4.33

A-20 5.00 4.50 5.00 4.83

A-21 4.50 3.00 4.00 3.83

A-22 5.50 3.25 4.00 4.25

A-23 4.00 5.50 3.50 4.33

A-24 6.50 9.50 8.00 8.00

A-25 8.00 8.50 8.50 8.33

A-26 4.50 5.50 6.00 5.33

A-27 6.50 8.00 6.50 7.00

A-28 7.50 6.50 9.00 7.67

A-29 5.50 3.00 4.00 4.17

A-30 4.00 4.50 3.00 3.83

A-31 5.00 4.00 3.50 4.17

A-32 2.50 3.50 3.00 3.00

A-33 4.00 2.75 4.50 3.75


HPTLC Analysis

The fingerprints of the test solutions prepared from the flowers of all commercial marshmallow materials as well as the flower, corolla and calyx parts of reference Alcea sp. sample were compared (Figure 3, 4, 5, 6 and 7). Under 366 nm, A-4, 5, 9, 11, 12, 13, 17, 18, 19, 21, 23, 24, 25 and 32 showed two blue fluorescent zones and red fluorescent spot in the upper part of plate. Moreover, blue fluorescent zones at Rf 0.5 and 0.9 were seen in A-8, 10, 12, 14, 18, 20, 22, 28, 29, 30, 31 and 33. Furthermore, this blue zone was also detected in the flower, corolla and calyx parts of Alcea sp. Besides, after spraying with H2SO4 reagent, under white light, blackish yellow zone between Rf 0.2 and 0.4 was observed in A-4, 5, 9, 11, 12, 13, 17, 18, 19, 24, 25 and 32. In addition, they exhibited a dark yellow zone at Rf 0.8, so they were similar to each other.

Figure 3. HPTLC chromatogram-1 visualized (a) at 366 nm; (b) under white light. Mobile phase: acetic acid-water-butanol (15:30:60, v/v/v); applied volumes: Mallow-7 μL, other extracts-9 μL, derivatization: H2SO4 reagent. Tracks: (1) Alcea sp. (reference); (2) mallow; (3) A-15; (4) A-1; (5) A-2; (6) A-3; (7) A-4; (8) A-5; (9) A-6; (10) A-7; (11) A-8; (12) A-9.


Figure 4. HPTLC chromatogram-2 visualized (a) at 366 nm; (b) under white light. Mobile phase: acetic acid-water-butanol (15:30:60, v/v/v), applied volumes: Mallow-7 μL, other ex-tracts-9 μL, derivatization: H2SO4 reagent. Tracks: (1) Alcea sp. (reference); (2) mallow; (3) A-10; (4) A-11; (5) A-12; (6) A-13; (7) A-14; (8) A-16; (9) A-17; (10) A-18; (11) A-19; (12) A-20.

Figure 5. HPTLC chromatogram-3 visualized (a) at 366 nm; (b) under white light. Mobile phase: acetic acid-water-butanol (15:30:60, v/v/v), applied volumes: Mallow-7 μL, other extracts-9 μL, derivatization: H2SO4 reagent. Tracks: (1) Alcea sp. (reference); (2) mallow; (3) A-21; (4) A-22; (5) A-23; (6) A-24; (7) A-25.


Figure 6. HPTLC chromatogram-4 visualized (a) at 366 nm; (b) under white light. Mobile phase: acetic acid-water-butanol (15:30:60, v/v/v), applied volumes: Mallow-7 μL, other extracts-9 μL, derivatization: H2SO4 reagent. Tracks: (1) Alcea sp. (reference); (2) Mallow; (3) A-26; (4) A-27; (5) A-28; (6) A-29; (7) A-30; (8) A-31; (9) A-32; (10) A-33; (11) A-5.

Figure 7. HPTLC chromatogram-5 visualized (a) at 366 nm; (b) under white light. Mobile phase: acetic acid-water-butanol (15:30:60, v/v/v), applied volumes: Mallow-7 μL, other extracts-9 μL, derivatization: H2SO4 reagent. Tracks: (1) Alcea sp. whole flower (reference); (2) Mallow flower; (3) Alcea sp. corolla; (4) Alcea sp. calyx.


DISCUSSION

In the present study, a monograph for “Althaeae flos” was proposed following the template for “Malvae sylvestris flos” in Ph. Eur. to be used in determination of the qualification of marshmallow flower samples and the developed template was applied for quality assessment of commercial marshmallow flower samples sold in herbal stores under the name of “hatmi çiçeği”.

Macroscopic (Table 2) and microscopic (Table 3) examinations revealed that all commercial marshmallow samples had quite similar morphological and micro-scopic properties to those itemized under “Malvae sylvestris flos” monograph [Ph. Eur.]. In addition, it was observed that six samples had stomata with three sub-sidiary cells, and only one sample had Malvaceae type glandular trichome. On the other hand, it was found that petal lengths of all commercial marshmallow sam-ples were longer than the specified values for Althaea species [9-16 mm]1, but were similar to those issued for Alcea species [30 mm or longer].39 Besides, two-locular ovary was observed in all marshmallow materials. As a result, it was considered that all commercial marshmallow samples were originally obtained from Alcea sp. flowers, but not from the official species Althaea officinalis. This is possibly due to widespread distribution of Alcea species in Turkey comparing to Althaea species.

In foreign matter analysis, only five samples were considered to be acceptable since they were free from insects or snails residues as well as the ratio of foreign elements were found to be lower than the set value 2.0% (Table 4). On the other hand, 80% of the commercial marshmallow samples were found to be contami-nated with insects, flies, snails and other animal wastes. The possible reason why these materials contain lots of such unacceptable matters is the closure of the petals during harvesting of the flowers, and the insects or flies collecting nectar from the flowers confined inside the closed flowers. Under these circumstances, it is not possible to prohibit such foreign substances without applying insecticide to the flowers, which would increase the health risk. For this reason, in contrast to Althaea or Alcea species, use of Malva sylvestris flowers does not have a risk for such contaminations and may be safely used.

In loss on drying test, it was detected that the rates of five commercial marsh-mallow samples and Alcea sp. were found to be more than 12.0%, but the rates in the remaining 28 marshmallow materials were below this ratio. In total ash analysis, the ratio of all commercial marshmallow materials and Alcea sp. were found to be lower than 14.0%. In swelling index test, it was determined that the results of all marshmallow materials and Alcea sp. were below the set value of 15 (Table 5), thus it was revealed that the mucilage content in marshmallow flowers was less than that in mallow flower.


In HPTLC analysis (Figure 3 to Figure 7), when the chromatographic profiles at 366 nm were compared, it was determined that fourteen marshmallow samples presented 2 blue fluorescent zones and a red fluorescent zone in the upper part. In addition, blue fluorescent zones at Rf 0.5 and 0.9 were found in the chroma-togram of twelve samples. This blue zone was also detected in the flower, corolla and calyx parts of Alcea sp. Furthermore, after spraying with H2SO4 reagent, under white light, blackish yellow spot between Rf 0.2 and 0.4 as well as dark yellow zone at Rf 0.8 were observed in twelve specimens.

CONCLUSION

Although the roots (Althaeae radix) and leaves (Althaeae folium) of Althaea offici-nalis are the officially accepted herbal drugs in Ph. Eur., only its flowers are avail-able in the herbal markets in Turkey. In this study, primarily a draft monograph for the flowers was prepared in order to set its specifications. Then the commer-cial flower samples which are sold under the name of “hatmi çiçeği” in the herbal markets were analyzed for their compliance to the specifications set. These results have revealed that all the available “hatmi” flowers samples were obtained from Alcea sp., but not from Althaea. On the other hand, due to insect and snails parts as well as high ratio of foreign matter, including feathers, bristles, stones and for-eign plant parts, which were detected in the flower samples in 28 out of 33 samples obtained from herbal markets, they were rated out of official use.

In the light of the discussed above, a template for Althaeae flos monograph is prepared as follows:

The values in the experimental sections of the “Althaeae flos” monograph were the results obtained from the tests carried out in this study. The ratio of foreign matter was found to be between 0.029 and 5.696%. The mean calculated for 33 marshmallow samples was found to be 1.13. Maximum 2.0 per cent was approved as the amount of foreign matter by predicating on the general pharmacopeia rule.

In loss on drying test, the rates were between 9.74 and 13.25%. The average of 33 marshmallow materials was found to be 10.99. When “Malvae sylvestris flos” monograph [Ph. Eur.] value and the average of findings were considered, maxi-mum 12.0% was accepted as the quantity of loss on drying in the “Althaeae flos” monograph.

In total ash experiment, the lowest rate was 8.36%, and the highest one was 13.52%. The mean of 33 marshmallow materials was found to be 10.24. When the results were taken into consideration, maximum 12.0% was approved as the content of total ash in the “Althaeae flos” monograph.

In swelling index trial, the lowest value was 2.75, and the highest value was 8.33.


The average of 33 marshmallow samples was 4.94. Regarding to the findings of trials, minimum 7 was accepted as the value of swelling index in the “Althaeae flos” monograph.

In TLC analyses, marshmallow specimens showed blue fluorescent spot and red fluorescent spot in the upper part of the chromatogram that was similar to Malvae sylvestris flos test solution [Ph. Eur.]. Considering the findings and TLC results stated in the “Malvae sylvestris flos” monograph, it is accepted that test solutions of marshmallow flowers exhibited a red spot in the upper part of the middle third and violet zone in the middle third.

According to the findings obtained from the Ph. Eur. analyses applied to all the samples and the evaluations of literature data, an “Althaeae flos” monograph prepared in Ph. Eur. template is as follows.

MARSHMALLOW FLOWER

Althaeae flos

DEFINITION

Whole or fragmented dried flower of Althaea officinalis L. and Alcea species.

IDENTIFICATION

A. Flowers borne in fascicles in the leaf axils. The flower involves an epicalyx with 6-9 lanceolate segments, connate at the base and located below the calyx. A calyx with 5 pubescent triangular lobes, gamosepalous at the base, not inflated. A corolla with 5 petals white to pinkish white, fused to the staminal tube at the base. Style much branched at the apex. Althaea petals 9-14 mm, Alcea petals 30 mm or more. Stamens numerous, the filaments of which fuse into a stami-nal tube covered by simple trichomes and small stellate trichomes visible using a lens. Althaea anthers brownish purple, Alcea anthers yellowish. Carpels nu-merous, glabrous, in one series around the base of the styles, 1-ovulate. Althaea carpels unilocular, Alcea carpels sub-bilocular. Fruit a schizocarp, splitting into numerous 1-seeded, indehiscent mericarps. Mericarps rugose, stellate-pilose.

B. Reduce to a powder (355) (2.9.12). The powder is pinkish white. Examine under a microscope using chloral hydrate solution R. The powder displays the following diagnostic characters (Figure 8): unicellular, thick-walled trichomes; small unicellular covering trichomes, somewhat curved [A] or in star-shaped groups of 2-9 [B]; capitate glandular trichomes with multicellular heads [C]; mesophyll fragments with cluster crystals of calcium oxalate [D]; veins of sepals with vessel [E]; fragments of petal epidermis [F] accompanied by glandular tri-chomes with multicellular heads [G]; numerous spherical pollen grains with a spiny exine, 140-160 µm in diameter [H].


Figure 8. Illustration for identification test B of powdered herbal flower drug: (A) curved trichome; (B) stellate trichome; (C) glandular trichome; (D) mesophyll fragments with cluster crystals of calcium oxalate; (E) vessel; (F) petal epidermis; (G) petal epidermis accompanied by glandular trichome with multicellular heads; (E) pollen grain.


C. Thin-layer chromatography (2.2.27).

Test solution. To 1 g of the pulverized drug (355) (2.9.12) add 10 mL of ethanol (60 per cent, v/v) R. Stir for 15 min and filter.

Plate: TLC silica gel plate R.

Mobile phase: acetic acid R, water R, butanol R (15:30:60, v/v/v).

Application: 9 mL of test solution, as 9.0 mm bands.

Development: over a path of 10 cm.

Drying: in air.

Detection: examine in daylight, ultraviolet light at 366 nm, spray with sulphuric acid solution (5 per cent) and exsiccate in an oven at 105°C.

Results: chromatogram attained with test solution exhibits red zone in the upper part of the middle third and violet zone in the middle third.

TESTS

Foreign matter (2.2.32): maximum 2.0 per cent

Loss on drying (2.4.16): maximum 12.0 per cent, detected on 1.000 g of the pulverized herbal drug by desiccating in an oven at 105°C for 2 hours.

Total ash (2.8.1): maximum 12.0 per cent.

Swelling index (2.8.4): minimum 7, detected on 0.2 g of the pulverized herbal drug (710) (2.9.12) moistened with 0.5 mL of ethanol R.

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